Origin of the multi-phasic quenching dynamics in the BLUF domains across the species

Blue light using flavin (BLUF) photoreceptors respond to light via one of nature’s smallest photo-switching domains. Upon photo-activation, the flavin cofactor in the BLUF domain exhibits multi-phasic dynamics, quenched by a proton-coupled electron transfer reaction involving the conserved Tyr and Gln. The dynamic behavior varies drastically across different species, the origin of which remains controversial. Here, we incorporate site-specific fluorinated Trp into three BLUF proteins, i.e., AppA, OaPAC and SyPixD, and characterize the percentages for the Wout, WinNHin and WinNHout conformations using 19F nuclear magnetic resonance spectroscopy. Using femtosecond spectroscopy, we identify that one key WinNHin conformation can introduce a branching one-step proton transfer in AppA and a two-step proton transfer in OaPAC and SyPixD. Correlating the flavin quenching dynamics with the active-site structural heterogeneity, we conclude that the quenching rate is determined by the percentage of WinNHin, which encodes a Tyr-Gln configuration that is not conducive to proton transfer.


Data Policy information about availability of data
All manuscripts must include a data availability statement.This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy

Human research participants
Policy information about studies involving human research participants and Sex and Gender in Research.
Reporting on sex and gender

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Field-specific reporting
Please select the one below that is the best fit for your research.If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Blinding
Behavioural & social sciences study design All studies must disclose on these points even when the disclosure is negative.

Research sample
The transient absorption experimental data and NMR experimental data have been deposited in the figshare database under accession code [https:// doi.org/10.6084/m9.figshare.24441943].The BLUF structures used in this study were obtained from the Protein Data Bank (PDB) with accession codes 1YRX and 2HFO.Source data are provided as a Source Data file with this paper.
No sample size calculations were performed.Sample size was determined to be adequate based on the magnitude and consistency of measurable differences between groups.The sample sizes used for data averaging were sufficient for data analysis.
No data was excluded.
Transient absorption experiment and NMR experiemnt for each sample were repeated three times, and we confirm the consistency of the results.The final dataset is averaged data from the repeated experimental data.
No randomization was applied in this study, since our work does not concern large number of statistical data points.
Blinding was not relevant in this study, since our work does not concern large number of statistical data points.
Briefly describe the study type including whether data are quantitative, qualitative, or mixed-methods (e.g.qualitative cross-sectional, quantitative experimental, mixed-methods case study Provide details about the data collection procedure, including the instruments or devices used to record the data (e.g.pen and paper, computer, eye tracker, video or audio equipment) whether anyone was present besides the participant(s) and the researcher, and whether the researcher was blind to experimental condition and/or the study hypothesis during data collection.
Indicate the start and stop dates of data collection.If there is a gap between collection periods, state the dates for each sample cohort.
If no data were excluded from the analyses, state so OR if data were excluded, provide the exact number of exclusions and the rationale behind them, indicating whether exclusion criteria were pre-established.
State how many participants dropped out/declined participation and the reason(s) given OR provide response rate OR state that no participants dropped out/declined participation.
If participants were not allocated into experimental groups, state so OR describe how participants were allocated to groups, and if allocation was not random, describe how covariates were controlled.
Briefly describe the study.For quantitative data include treatment factors and interactions, design structure (e.g.factorial, nested, hierarchical), nature and number of experimental units and replicates.
Describe the research sample (e.g. a group of tagged Passer domesticus, all Stenocereus thurberi within Organ Pipe Cactus National Monument), and provide a rationale for the sample choice.When relevant, describe the organism taxa, source, sex, age range and any manipulations.State what population the sample is meant to represent when applicable.For studies involving existing datasets, describe the data and its source.
Note the sampling procedure.Describe the statistical methods that were used to predetermine sample size OR if no sample-size calculation was performed, describe how sample sizes were chosen and provide a rationale for why these sample sizes are sufficient.
Describe the data collection procedure, including who recorded the data and how.
Indicate the start and stop dates of data collection, noting the frequency and periodicity of sampling and providing a rationale for these choices.If there is a gap between collection periods, state the dates for each sample cohort.Specify the spatial scale from which the data are taken If no data were excluded from the analyses, state so OR if data were excluded, describe the exclusions and the rationale behind them, indicating whether exclusion criteria were pre-established.
Describe the measures taken to verify the reproducibility of experimental findings.For each experiment, note whether any attempts to repeat the experiment failed OR state that all attempts to repeat the experiment were successful.
Describe how samples/organisms/participants were allocated into groups.If allocation was not random, describe how covariates were controlled.If this is not relevant to your study, explain why.
Describe the extent of blinding used during data acquisition and analysis.If blinding was not possible, describe why OR explain why blinding was not relevant to your study.
Describe the study conditions for field work, providing relevant parameters (e.g.temperature, rainfall).
State the location of the sampling or experiment, providing relevant parameters (e.g.latitude and longitude, elevation, water depth).
Describe the efforts you have made to access habitats and to collect and import/export your samples in a responsible manner and in compliance with local, national and international laws, noting any permits that were obtained (give the name of the issuing authority, the date of issue, and any identifying information).

nature portfolio | reporting summary
March 2021

Disturbance
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Ethics oversight
Note that full information on the approval of the study protocol must also be provided in the manuscript.
Describe any disturbance caused by the study and how it was minimized.
Describe all antibodies used in the study; as applicable, provide supplier name, catalog number, clone name, and lot number.
Describe the validation of each primary antibody for the species and application, noting any validation statements on the manufacturer's website, relevant citations, antibody profiles in online databases, or data provided in the manuscript.
State the source of each cell line used and the sex of all primary cell lines and cells derived from human participants or vertebrate models.
Describe the authentication procedures for each cell line used OR declare that none of the cell lines used were authenticated.
Confirm that all cell lines tested negative for mycoplasma contamination OR describe the results of the testing for mycoplasma contamination OR declare that the cell lines were not tested for mycoplasma contamination.
Name any commonly misidentified cell lines used in the study and provide a rationale for their use.
Provide provenance information for specimens and describe permits that were obtained for the work (including the name of the issuing authority, the date of issue, and any identifying information).Permits should encompass collection and, where applicable, export.
Indicate where the specimens have been deposited to permit free access by other researchers.
If new dates are provided, describe how they were obtained (e.g.collection, storage, sample pretreatment and measurement), where they were obtained (i.e.lab name), the calibration program and the protocol for quality assurance OR state that no new dates are provided.
Identify the organization(s) that approved or provided guidance on the study protocol, OR state that no ethical approval or guidance was required and explain why not.

ChIP-seq Data deposition
Confirm that both raw and final processed data have been deposited in a public database such as GEO.
Confirm that you have deposited or provided access to graph files (e.g.BED files) for the called peaks.

Data access links
May remain private before publication.

Files in database submission
Genome browser session The axis labels state the marker and fluorochrome used (e.g.CD4-FITC).
The axis scales are clearly visible.Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Instrument
For "Initial submission" or "Revised version" documents, provide reviewer access links.For your "Final submission" document, provide a link to the deposited data.
Provide a list of all files available in the database submission.
Provide a link to an anonymized genome browser session for "Initial submission" and "Revised version" documents only, to enable peer review.Write "no longer applicable" for "Final submission" documents.
Describe the experimental replicates, specifying number, type and replicate agreement.
Describe the sequencing depth for each experiment, providing the total number of reads, uniquely mapped reads, length of reads and whether they were paired-or single-end.
Describe the antibodies used for the ChIP-seq experiments; as applicable, provide supplier name, catalog number, clone name, and lot number.
Specify the command line program and parameters used for read mapping and peak calling, including the ChIP, control and index files used.
Describe the methods used to ensure data quality in full detail, including how many peaks are at FDR 5% and above 5-fold enrichment.
Describe the software used to collect and analyze the ChIP-seq data.For custom code that has been deposited into a community repository, provide accession details.
Describe the sample preparation, detailing the biological source of the cells and any tissue processing steps used.
Identify the instrument used for data collection, specifying make and model number.

Correction
Describe the software used to collect and analyze the flow cytometry data.For custom code that has been deposited into a community repository, provide accession details.
Describe the abundance of the relevant cell populations within post-sort fractions, providing details on the purity of the samples and how it was determined.
Describe the gating strategy used for all relevant experiments, specifying the preliminary FSC/SSC gates of the starting cell population, indicating where boundaries between "positive" and "negative" staining cell populations are defined.
Indicate task or resting state; event-related or block design.
Specify the number of blocks, trials or experimental units per session and/or subject, and specify the length of each trial or block (if trials are blocked) and interval between trials.
State number and/or type of variables recorded (e.g.correct button press, response time) and what statistics were used to establish that the subjects were performing the task as expected (e.g.mean, range, and/or standard deviation across subjects).

Specify in Tesla
Specify the pulse sequence type (gradient echo, spin echo, etc.), imaging type (EPI, spiral, etc.), field of view, matrix size, slice thickness, orientation and TE/TR/flip angle.
State whether a whole brain scan was used OR define the area of acquisition, describing how the region was determined.
Provide detail on software version and revision number and on specific parameters (model/functions, brain extraction, segmentation, smoothing kernel size, etc.).
If data were normalized/standardized, describe the approach(es): specify linear or non-linear and define image types used for transformation OR indicate that data were not normalized and explain rationale for lack of normalization.
Describe the template used for normalization/transformation, specifying subject space or group standardized space (e.g.original Talairach, MNI305, ICBM152) OR indicate that the data were not normalized.
Describe your procedure(s) for artifact and structured noise removal, specifying motion parameters, tissue signals and physiological signals (heart rate, respiration).
Define your software and/or method and criteria for volume censoring, and state the extent of such censoring.
Specify type (mass univariate, multivariate, RSA, predictive, etc.) and describe essential details of the model at the first and second levels (e.g.fixed, random or mixed effects; drift or auto-correlation).
Define precise effect in terms of the task or stimulus conditions instead of psychological concepts and indicate whether ANOVA or factorial designs were used.
Specify voxel-wise or cluster-wise and report all relevant parameters for cluster-wise methods.
Describe the type of correction and how it is obtained for multiple comparisons (e.g.FWE, FDR, permutation or Monte Carlo).
to confirm that the raw and calibrated dates are available in the paper or in Supplementary Information.
involve any of these experiments of concern: No Yes Demonstrate how to render a vaccine ineffective Confer resistance to therapeutically useful antibiotics or antiviral agents Enhance the virulence of a pathogen or render a nonpathogen virulent Increase transmissibility of a pathogen Alter the host range of a pathogen Enable evasion of diagnostic/detection modalities Enable the weaponization of a biological agent or toxin Any other potentially harmful combination of experiments and agents Provide a rationale for the study sample chosen.For studies involving existing datasets, please describe the dataset and source.Describe the sampling procedure (e.g.random, snowball, stratified, convenience).Describe the statistical methods that were used to predetermine sample size OR if no sample-size calculation was performed, describe how sample sizes were chosen and provide a rationale for why these sample sizes are sufficient.For qualitative data, please indicate whether data saturation was considered, and what criteria were used to decide that no further sampling was needed.
).State the research sample (e.g.Harvard university undergraduates, villagers in rural India) and provide relevant demographic information (e.g.age, sex) and indicate whether the sample is representative.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.